1 research outputs found
Rapid and Sensitive Genotyping of SARS-CoV‑2 Key Mutation L452R with an RPA-<i>Pf</i>Ago Method
In the two years of COVID-19 pandemic, the SARS-CoV-2
variants
have caused waves of infections one after another, and the pandemic
is not ending. The key mutations on the S protein enable the variants
with enhanced viral infectivity, immune evasion, and/or antibody neutralization
resistance, bringing difficulties to epidemic prevention and control.
In support of precise epidemic control and precision medicine of the
virus, a fast and simple genotyping method for the key mutations of
SARS-CoV-2 variants needs to be developed. By utilizing the specific
recognition and cleavage property of the nuclease Argonaute from Pyrococcus furiosus (PfAgo), we developed
a recombinase polymerase amplification (RPA) and PfAgo combined method for a rapid and sensitive genotyping of SARS-CoV-2
key mutation L452R. With a delicate design of the strategy, careful
screening of the RPA primers and PfAgo gDNA, and
optimization of the reaction, the method achieves a high sensitivity
of a single copy per reaction, which is validated with the pseudovirus.
This is the highest sensitivity that can be achieved theoretically
and the highest sensitivity as compared to the available SARS-CoV-2
genotyping assays. Using RPA, the procedure of the method is finished
within 1.5 h and only needs a minimum laboratorial support, suggesting
that the method can be easily applied locally or on-site. The RPA-PfAgo method established in this study provides a strong
support to the precise epidemic control and precision medicine of
SARS-CoV-2 variants and can be readily developed for the simultaneous
genotyping of multiple SARS-CoV-2 mutations